(1) Field of the Invention
The present invention relates to the enzyme alkaline phosphatase and more particularly to this enzyme derived from Pandalus borealis and to nucleic acid molecules encoding it.
(2) Description of Related Art
Alkaline phosphatases (E.C. 3.1.3.1), having the alternative names alkaline phosphomonoesterase, phosphomonoesterase or glycerophosphatase, are orthophosphoric-monoester phosphohydrolases with enzyme activity optima at alkaline conditions. Examples of alkaline phosphatase (ALP) substrates are DNA, RNA, and ribo-as well as deoxyribonucleoside triphosphates. The hydrolysis produces an alcohol and an orthophosphate. In other words, ALPs dephosphorylate DNA, RNA, rNTPs and dNTPs. Dephosphorylation of protein by various ALPs have also been reported. ALPs are widespread in nature, found in organisms ranging from bacteria to humans. Complex organisms usually contain both tissue-specific and non-specific ALPs. The polypeptides differ in size from 15 to 170 kDa. Some of these proteins are bound or “anchored” to cellular membranes. Most commonly the enzymes have a requirement for divalent metal cations such as Mg2+ or Zn2+.
Current usage of ALPs in molecular biology is focused on, but not limited to, three main applications of DNA analysis or preparation: 1) dephosphorylation of vector DNA after restriction enzyme digestions to minimise self-ligation of the cloning vector, thus favouring ligation of insert to the vector and creating a recombinant construct, 2) dephosphorylation of dNTPs after PCR amplifications, in combined use with a single-strand exonuclease that hydrolyses primers to dNTPs, to omit the need of further clean-up before direct DNA sequencing of PCR products, or 3) dephosphorylation of DNA ends for subsequent labelling with 32P using [γ-32P] NTP and T4 polynucleotide kinase. The described ALP enzyme reactions are intermediate steps in DNA analysis processes. Other important applications known in the art include those where the enzyme activities are used as reporters such as in enzyme-linked immunosorbent assay (ELISA), in gene-fusion or gene-delivery systems, or in conjugation to oligonucleotides used as hybridisation probes.
Three main ALPs are used in commercially available products;                i) calf intestinal alkaline phosphatase (CIAP)        ii) shrimp alkaline phosphatase (SAP) from the arctic shrimp Pandalus borealis         iii) bacterial alkaline phosphatase (BAP)        
The animal CIAP and SAP enzymes have similar specific activities of 2000-4000 units/mg protein compared to 50 units/mg BAP protein, which make the two former enzymes more attractive as efficient enzymes. The SAP enzyme is inactivated by moderate heat as discussed below, and is thus preferred for several applications in which the enzyme activity needs to be removed prior to further steps in the processes.
A genetically engineered temperature sensitive BAP mutant was reported [Shandilya, H. And Chatterjee, D. K., 1995. An engineered thermosensitive alkaline phosphatase for dephosphorylating DNA. Focus, 17 (3): 93-95] with no details given to describe the engineering. This mutant enzyme (TsAP), sold by LifeTechnologies, Inc., is inactivated (95% or more) by heat (65° C. for 15 min) in the presence of EDTA only. The recommended reaction temperature for the mutant and the wild-type enzyme is also 65° C. TsAP has at least 40-fold higher activity than wild-type BAP. With reference to high specific activity, TsAP is almost comparable to other ALPs such as CIAP and SAP.
Two heat-labile ALPs from a psycrophilic microorganism have been purified and characterised [de Prada, P., and Brenchley, J. E., 1997, Purification and characterization of two extracellular alkaline phosphatases from a psycrophilic Arthrobacter isolate, Appl. Env. Microbiol., 63(7): 2928-2931]. The enzymes that varied with respect to substrate specificities and kinetic properties, displayed different heat-labilities of which the most labile resembles the SAP enzyme lability. No specific activity in units/mg protein and no primary structures were reported.
A cold-adapted ALP from atlantic cod was isolated and characterised [Ásgeirsson, B., Hartemink, R., and Chlebowski, J. F., 1995, Alkaline phosphatase from atlantic cod (Gadus morhua). Kinetic and structural properties which indicate adaption to low temperatures. Comp. Biochem. Physiol., 110B(2): 315-329]. The enzyme showed thermolability similar to SAP. No primary structure of the protein/gene was provided.
A study on ALP isozymes from trout [Whitmore, D. H., and Goldberg, E., 1972, Trout intestinal alkaline phosphatases II. The effect of temperature upon enzymatic activity in vitro and in vivo. J. Exp. Zool., 182: 59-68] showed that temperatures of the environment affects the isozyme pattern, and that some isoforms are thermolabile.
Shrimps from the warm water region outside Taiwan contain several ALPs [Lee, A.-C., and Chuang, N.-N., 1991, Charaterization of different molecular forms of alkaline phosphatase in the hepatopancreas from shrimp Penaeus monodon (Crustacea: Depacoda). Comp. Biochem. Physiol., 99B(4): 845-850], and the enzymes were concluded to be heat-stable. No primary structures were provided.
An alkaline phosphatase activity from the hepatopancreas of arctic shrimp Pandalus borealis was found to be contained in the processing wastewater from the shrimp industry [Olsen, R. L., Johansen, A., and Myrnes, B., 1990, Recovery of enzymes from shrimp waste. Process Biochem. 25:67-68], and the enzyme was later purified from the hepatopancreas [Olsen, R. L., Øverbø, K., and Myrnes, B., 1991, Alkaline phosphatase from hepatopancreas of shrimp (Pandalus borealis): a dimeric enzyme with catalytically active subunits. Comp. Biochem. Physiol. 99B(4):755-761]. This purified protein had an apparent molecular weight of 65 kDa (each subunit) and was shown to be a dimeric enzyme with catalytically active subunits in contrast to most other animal ALPs that require dimerisation for activity. According to the report SAP has an isoelectric point of 3.7. The shrimp enzyme efficiently removes terminal 5′ phosphate from any DNA strand termini (5′ and 3′ overhang or blunt ends) produced by restriction endonucleases, although 5′ protruding ends are more reactive than blunt ends or 5′ recessive ends.
Relative to CIAP, the SAP enzyme has a slight shift to lower temperatures for activity, but it is not considered to be truly cold-active. With a maximum enzyme activity at about 40° C. (45° C. for CIAP), almost 40% activity is retained at 10° C. or at 50° C. compared to 10% and 90% activity, respectively, for CIAP. Although the temperature for maximum activity is close to 40° C., the SAP enzyme starts to loose activity when pre-incubated for a period of 15 min beyond 37° C. After pre-incubation at 65° C. the SAP activity is reduced by 95% or more and the activity is undetectable after pre-incubation at 70° C. In comparison, after similar heat-treatments CIAP retains 40% and 20% of its activity.
Thus, relative to its commercial competitor, the SAP enzyme is heat-labile and cold-active making it particularly suited for use in multi-step laboratory protocols where a simple heating step can de-activate the enzyme so that it plays no part in further method steps.
BIOTEC ASA, Tromsø, Norway, produces the commercial SAP enzyme, from the shrimp industry wastewater. Onboard the trawlers, freshly collected shrimps are frozen in large blocks. When landed the shrimps are carefully thawed by re-circulated cold water. Approximately 1000 l of water is used for 4000 kg of shrimp. During the process of freezing and thawing, the shrimp hepatopancreas breaks and the contents are released to the water. This wastewater is then concentrated and several chromatographic steps are used to purify the SAP enzyme.
The producer supplies SAP for the world market through well-known companies like USB, Boehringer-Mannheim or Amersham Pharmacia Biotech.
Due to its high specific activity, its “versatility” regarding DNA termini, and its relative temperature-lability, SAP is frequently used in dephosphorylation of cloning vectors prior to ligation reactions, and in treatment of PCR amplification product-mixtures prior to DNA sequencing reactions as described in U.S. Pat. Nos. 5,741,676 and 5,756,285.
The present production of SAP suffers from varying quality of the wastewater, which again affects the production efficiency. Two factors cause this variation; i) natural seasonal variation of enzyme production in the shrimp; and ii) the handling of the shrimp source prior to or during freezing and the handling of shrimps or water during or after the thawing process.
There is also a concern about the future availability of wastewater; i) as a natural resource shrimps are not guaranteed to be available at all times; and ii) the shrimp industry is now looking into possible new ways to freeze the shrimps, i.e. single-freezing, from which the wastewater has been tested to contain small amounts of enzymes such as SAP.
In addition to these practical problems, the market has a demand for a recombinant SAP product as recombinant products are frequently preferred in molecular biology techniques, particularly where product purity is an issue, e.g. in the production of DNA based therapeutics or in forensic science. SAP has highly advantageous enzymatic and physiochemical properties and is a preferred enzyme for the laboratory protocols in which it is useful. There is therefore an appreciable need for a synthetic or recombinant source of SAP, which is produced in a uniform and pure fashion. However, neither DNA or amino acid sequences for SAP have been previously elucidated.
In order to isolate the gene and to subsequently clone and produce a recombinant SAP, several attempts have been made to obtain an N-terminal sequence of the purified protein. None of these attempts have succeeded. The sequence analyses have revealed multiple (four or more) alternative amino acids for each specific N-terminal position. Thus, no protein sequence information has been available for use, even with highly degenerate oligonucleotide probes, to hunt for the SAP gene. The reason(s) for the non-homologous N-termini in an apparently homogenous enzyme preparation can only be speculated. It is possible that SAP isoforms are produced by different genes, by alternative splicing or by varying post-translational modifications of the protein, or that SAP is attacked by proteases at the N-terminus with no detectable reduction in molecular weight.
It has also been found by the present inventors that the limited homology between the alkaline phosphatase from the hepatopancreas of arctic shrimps (Pandalus borealis) and alkaline phosphatase from species which have been sequenced prevents the routine isolation of nucleic acid sequences encoding alkaline phosphatase from Pandalus borealis. 